Pancreatic lipase analysis, which is useful for pancreatic disease diagnosis, is often carried out by measuring pancreatic lipase under conditions in which a micellar substrate is dispersed in water in view of the fact that pancreatic lipase functions in oil-water interface. This is because non-pancreatic lipase such as lipoprotein lipase or esterase reacts with a substrate solubilized with a surfactant or the like or with glyceride comprising fatty acid having short alkyl chains. Thus, it is considered that a technically important point for development of dry analytical elements for pancreatic lipase is the incorporation of a long-fatty-acid glyceride serving as a substrate, which is specific to pancreatic lipase, into such analytical element, provided that glyceride is in a state such that it is specific to pancreatic lipase.
Dry analytical elements used for lipase analysis are roughly classified into two types. An example of a first type is a dry analytical element obtained by a method using 1,2-O-dilauryl-rac-glycero-3-glytaric acid/resorufin ester serving as a dye-releasing substrate (JP Patent Publication (Kokai) No. 9-154598 A (1997)). Such method is preferable because high specificity with respect to pancreatic lipase can be achieved and a glycerin coloring system is unnecessary. However, such substrate incorporated into a dry analytical element is highly likely to disintegrate. Thus, such dry analytical element has still not been available in practice, although it has been attempted to separate a low-pH layer containing a lipase substrate from another high-pH reagent layer. In addition, the relatively high price of such a substrate is also problematic in terms of practical use.
An example of a second type is a dry analytical element for lipase analysis, in which a method for converting triglyceride used as a substrate into a dye via glycerin and hydrogen peroxide is used. According to the first disclosed method, it is a multilayer dry analytical element (JP Patent Publication (Kokai) No. 59-48098 A (1984)), wherein triglyceride having a long chain alkyl group having at least 8 carbon atoms at an ester position (α position) and two other esters each having a short chain alkyl group is used as a substrate, water-soluble 1,2 diacetylglyceride generated in the presence of lipase in a specimen is converted into glycerin with the use of an esterase (namely, acetinase), and glycerin is converted into a dye. The above method is a convenient and highly accurate lipase measurement method. However, it has been reported that selectivity with respect to pancreatic lipase is not high, and thus that attention is required if this method is applied to the diagnosis of pancreatic diseases [Clin. Chem., 37/3, 447-451 (1991)]. Such problem regarding specificity may be caused by the fact that triglyceride used as a substrate also contains a short chain alkyl group.
Next, there has been disclosed a method, which also uses triglyceride as a substrate. That is, there has been disclosed a dry chemistry reagent for pancreatic lipase analysis, which comprises triglyceride having only a long chain fatty acid containing 14 to 20 carbon atoms, such as triolein, as a substrate and which further comprises monoglyceride lipase and a glycerin measurement reagent (JP Patent Publication (Kokai) No. 4-316500 A (1992)). Such a method using triolein is anticipated to be highly specific to pancreatic lipase. However, in this triolein addition method, since a highly fat-soluble substrate is incorporated, a protective colloid such as gum Arabic is used to carry out aqueous emulsification dispersion involving an ultrasonic treatment (JP Patent Publication (Kokai) No. 4-316500 A (1992): Examples). In this emulsification dispersion method, it is necessary to maintain the reproducibility of substrate dispersion and uniformity in particle size distribution. Thus, it is thought that production by such method is difficult.
For instance, JP Patent Publication (Kokai) No. 4-316500 A (1992) contains the following description: “triglyceride, such as triolein, comprising a long chain fatty acid in each of three ester positions has the property of being emulsified with difficulty. Thus, even if a solution in which triolein has been uniformly emulsified and dispersed via agitation or by physical shearing force generated by ultrasound waves or the like is added in the presence of a surfactant or a protective colloid upon preparation of a dry reagent, water serving as a dispersion medium disappears when the reagent becomes dry, and thus an emulsified product aggregates or coalesces so as to adhere to the surface of a spreading layer, resulting in significant reduction in the surface area in oil-water interface. Upon measurement, even if a specimen (liquid) containing lipase is allowed to react with such dry reagent, triolein remains in a state of aggregating or coalescing and thus does not return to the original state of being dispersed because of lack of physical shearing force. The reaction field of lipase is an oil-water interface. Thus, a decrease in the surface area of an oil-water interface is thought to cause a decrease in reaction rate.”
In addition, in the examples of the method of JP Patent Publication (Kokai) No. 4-316500 A (1992), a filter and a nylon film is impregnated with a reagent. However, since a support is not used to maintain strength in the examples, it is considered difficult to carry out transportation and winding at a constant rate/high rate in the production process. Thus, in order to produce a dry analytical element having both measurement accuracy and productivity, addition of a support is almost essential.
A method modified from the method of JP Patent Publication (Kokai) No. 4-316500 A (1992), which comprises adding a support to produce a multilayer analytical element with high accuracy and incorporating fine particles therein so as to enhance lipase reactivity is described in JP Patent Publication (Kokai) No. 2002-125699 A. The applicant has further modified this method. The applicant has conceived of a method of adding glyceride, such as triolein, dissolved in an organic solvent such as ethanol to an analytical element comprising a support, thereby producing a dry analytical element that is highly specific to pancreatic lipase.
However, when such a dry analytical element comprising a support necessary for stable production and using triolein as a substrate was produced, unexpectedly, another serious problem occurred. That is to say, the following was found. That is, since triolein is oil, such triolein added to the reaction layer of lipase is easily transcribed on the back side of the support when the transported product is wound, and the transcribed triolein on the support is then transcribed on a pass roll used in transportation. The thus transcribed triolein reduces a friction between the pass roll necessary for transportation and the support, and transportation slip is thereby generated. In order to produce highly accurate dry analytical element, it is necessary to add a constant amount of reagent by coating, impregnation, etc. Generation of a slip during transportation makes addition of a constant amount of reagent impossible. This makes production of a dry analytical element for lipase measurement impossible. At the same time, it means that a producing apparatus having a transportation system, which has become contaminated by oil, cannot be used to produce products (e.g. a glucose analyzing device, a cholesterol analyzing device, etc.) used in highly accurate clinical analyses that require addition of a constant amount of reagent. Moreover, triolein transcribed on the support not only causes malfunction to the transportation system, but it also gives a positive error when it is transcribed on a device for measuring and analyzing neutral fats.
Due to the aforementioned problems, the product disclosed in JP Patent Publication (Kokai) No. 59-48098 A (1984) is still the only commercially available dry analytical element for lipase analysis, although the product has low pancreatic lipase specificity. Thus, dry analytical elements that are excellent in terms of reliability for diagnosis of pancreatic diseases have been awaited in the market.